1. Field of the Invention
The present invention relates to a method of lyophilizing ericoid mycorrhizal fungi, lyophilized ericoid mycorrhizal fungi per se, and a system for using the lyophilized ericoid mychorrhizal fungi, especially for micropropagated Ericaceous plants. The present invention also relates to any ericaceous plants propagated with the lyophilized ericoid fungi.
2. Description of the Related Art
Species in the Ericaceae are indigenous to almost every region of the world. These plants often thrive on otherwise unproductive sites because they develop specialized mycorrhizal associations common to most, if not all, ericaceous plants (Jackson and Mason, Mycorrhiza. Studies in Biology, no. 159, 1984). Many ericaceous plants are found on acidic, nutrient-poor soils high in organic matter. Association with an ericoid mycorrhizal fungus is often necessary for survival in such conditions. Ericoid mycorrhizal fungi can degrade organic residues produced by ericaceous plants themselves, thus providing mineral nutrients otherwise unavailable to their host plants (Read, In: Frontiers in Mycology: Honorary and General Lectures from the Fourth International Mycological Congress, Hawksworth-ed., 1990).
Micropropagation of ericaceous plants is a widely used method for propagation. Many media have been described to improve root development of the host plant and to increase the potential for synthesis of a mycorrhizal association between fungal inoculum and host. Moore-Parkhurst and Englander (Mycologia, Volume 73, 994-997, 1981) disclosed a method that included a paper bridge support over a liquid medium for synthesis of mycorrhizae between Hymnenoscyphus ericae and Rhododendron maximum (rosebay rhododendron). Similarly, Douglas et al. (Can. J. Bot., Volume 67, 2206-2212, 1989) disclosed the same system with a slightly modified liquid medium for inoculation of Rhododendron with Oidiodendron maius Barron. This method was effective in promoting growth of both plants and fungi. However, the procedure is very labor intensive and has limited commercial applications.
Pearson and Read (New Phytol., Volume 72, 371-379, 1973) developed a less complicated method for synthesis of ericoid mycorrhizae in aseptic culture. In this procedure, a layer of sterilized soil was placed on top of a water agar base which allowed a simple, effective method for promoting development of ericoid mycorrhizae with roots of host plants.
Hymenoscyphus ericae (Read) Korf and Kernan [syn. Pezizella ericae Read] is a widespread ericoid mycorrhizal fungus (Moore-Parkhurst and Englander, 1991, supra; Read, Can. J. Bot., Volluem 61, 381-419, 1983). Mycorrhizal associations with this fungus have been demonstrated with Calluna vulgaris (L.) Hull [heather, (Read, Trans. Brit. Mycol. Soc., Volume 63, 381-419, 1974)], Rhododendron chapmanii Gray [Chapman's rhododendron, (Barnes and Johnson, 1986, supra)], Rhododendron maximum L. (Duddridge and Read, Can. J. Bot., Volume 60, 2345-2356, 1982), Vaccinium angustrifolium Ait. [lowbush blueberry, (Couture et al., New Phytol., Volume 95, 315-380, 1983)] and V. corymobosum L. (Lareau, 1985, supra). Several fungi in the genus, Oidiodendron Robak, also form mycorrhizal associations with plants in the Ericaceae (Couture et al., 1983, supra; Currah et al., Can. J. Bot., Volume 71, 1481-1485, 1993, Dalpe, New Phytol., Volume 103, 391-396, 1986; Douglas et al., Can. J. Vot., Volume 67, 2206-2212, 1989).
Oidiodendron griseum Robak is a species that forms mycorrhizae with V. angustifolium (Couture, 1983, supra; Dalpe, supra) and V. corymbosum (Couture et al., 1983, supra; Lareau, 1985, supra). A related species, Oidiodendron maius Barron, was isolated from roots of Rhododendron X `Pink Pearl` and formed typical ericoid mycorrhizae when reassociated with rooted microsnoots of this cultivar (Douglas et al., 1989, supra). Other species of Oidiodendron forming ericoid mycorrhizae have been reported (Currah et al., 1993, supra; Dalpe, Can. J. Bot., Volume 69, 1712-1714, 1991; Stoyke and Currah, Can. J. Bot., Volume 69, 347-352, 1991; Xiao and Berch, Mycologia, Volume 84, 470-471, 1992).
A micropropagation protocol developed by Anderson (Proc. Intl. Plant Prop. Soc., Volume 28, 135-139, 1978) is currently used for rapid, commercial propagation of rhododendrons and includes an agar-solidified medium without addition of soil. A soilless medium may be used for direct rooting of microshoots following production of microshoots by this protocol, or microshoots may be induced to root in vitro in the agar medium. Micropropagated rhododendrons are often rooted ex vitro in a peat:vermiculite (V:V) medium (Barnes and Johnson, J. Environ. Hort., Volume 4, 109-111, 1986; Smagula and Litten, Acta Hort., volume 241, 110-114, 1989). However, Pieris D. Don. (andromeda) requires a root initiation phase in vitro (Pennell, The Plantsman, Volume 12, 120-125, 1990). Starrett et al. (J. Environ. Hort., Volume 11, 191-195, 1993) included an in vitro rooting step in a procedure developed for micropropagation of Pieris fioribunda (mountain andromeda).
Often, the greatest losses during micropropagation occur during plantlet acclimatization to greenhouse conditions (Preece and Sutter, In: Micropropagation, 71-93, Debergh and Zimmerman-ed., 1991). Typically, 10% of micropropagated plants in the Ericaceae either die or do not attain market standards during acclimatization, causing significant commercial losses (Lemoine et al., Agroniomie, Volume 12, 881-885, 1992). Unfortunately, ericaceous plants have exhibited mixed responses when inoculated with ericoid mycorrhizal fungi during micropropagation (Barnes and Johnson, 1986, supra; Berta and Gianinazzi-Pearson, In: Physiological and Genetical Aspects of Mycorrhizae, 673-676, Gianinazzi-Pearson and Gianinazzi-ed., 1986; Lareau, Acta Hort., volume 165, 197-205, 1985; Smagula and Litten, 1989, supra; Starrett et al., Proc. Southern Nurserymen's Assoc. Res. Conf., 40th Annu. Rpt., 266-268, 1995).
Fungal cultures are often preserved on fungal slants (Ainsworth, IN: Introduction to the History of Mycology, Cambridge University Press, New York, 359, 1976). Repeated transfers and long-term storage of active fungal cultures can result in physiological or morphological variation such as loss of pathogenicity. Inadvertent selection of atypical parts of a culture for transfer can also lead to stock cultures that differ from the orginal wild type. Risk of contamination and expense in labor and time requirements also increase with transfers of active cultures (Smith and Onions, The Preservation and Maintenance of Living Fungi. Commonwealth Mycol. Inst., Kew, Richmond, United Kingdom, 1983). Ectomycorrhizal fungi often die or lose their symbiotic ability after several years of repeated subculturing on agar media (Marx and Daniel, Can. J. Microbiol., Volume 22, 338-341, 1976).
A method of lyophilization of fungal cultures was proposed (Raper and Alexander, Mycologia, Volume 37, 499-525, 1945) to reduce losses common to transfer and storage. Long-term storage is especially important to preservation of mycorrhizal fungi. However, little information is available on potential applications of lyophilization for preservation of mycorrhizal fungi. Lyophilization has been effective only for species of arbuscular mycorrhizal fungi with thick-walled spores (Dalpe, IN: Proc. Seventh North Amer. Conf. on Mycorrhiza, 279, 1987, Sylvia et al.-eds.). Spores and hyphae of seven species of vesicular-arbuscular (VA) mycorrhizal fungi remained viable for eight years in dry storage in a vacuum (L-drying) (Tommerup, In: Proceedings of the 6th North American Conference on Mycorrhizae, 87, 1985). However, L-drying involves temperatures greater than zero degrees centegrade. Mature nonsporulating agar cultures of pathogenic fungi were preserved successfully by freeze-drying (Bazzigher, Phytopath. Z., Volume 45, 53-56, 1962). Some ascomycetous and basidiomycetous fungi survived lyophilization, however, cultures were revived immediately after lyophilization, and the long-term survival of freeze-dried cultures was not evaluated. A nonsporulating strain of Claviceps paspali Stev. and Hull. remained viable for 3 years following lyophilization (Pertot et al., Eur. J. Appl. Microbiol., Volume 4, 289-294, 1977). While there are various methods for lyophilizing and storing fungi, there remains a need in the art for a method for extended preservation of mycorrhizal fungi, especially nonsporulating ericoid mycorrhizal fungi, in order to attain long-term storage of viable fungi for use in a system to promote the survival of ericaceous plants, especially micropropagated ericaceous plants. The present invention is different from related art methods and provides lyophilized mycorrhizal fungi and uses for the lyophilized fungi.